Eukaryotes utilize a complex network to organize DNA, RNA, and proteins into cellular compartments. Despite their small size, spatial organization of proteins is also required for many bacterial processes, including adherence, cell division, and motility. Recent work has revealed a number of virulence factors that are targeted to specific locations in the bacterial cell, but the functional significance of subcellular protein localization for bacterial virulence has not been studied to date. However, the evolutionary conservation of such localization suggests that the localization of virulence factors within the cell during infection could impart significant advantages for bacterial pathogens. To establish a foundation for studying the role of protein localization in bacterial virulence, we are proposing two independent approaches that examine the organization of virulence determinants in the bacterium Pseudomonas aeruginosa. P. aeruginosa is an opportunistic pathogen that infects a wide variety of hosts and, in immuno-compromised individuals, leads to pneumonia, bacteremia, and eventually death. P. aeruginosa produces a single flagellum and multiple type IV pili that are required for adhesion and motility during infection of a host. The flagellum and pili are found only at the cell pole, representing excellent targets for monitoring discrete localization patterns in this bacterium. First, I will examine the mechanisms by which the flagellum and type IV pili become localized by characterizing the importance of candidate localization factors from other bacteria, such as the cytoskeletal elements MreB, MreC, and FtsZ. Excitingly, my preliminary results from this approach demonstrate a new role for MreB in localization and, potentially, function of type IV pili in P. aeruginosa. Second, I will perform a genetic screen for new factors important for localization of these structures. The genes involved in construction of the flagellum and pili are conserved amongst many bacteria, indicating that characterization of the factors governing their localization will provide insight into similar processes in other bacterial pathogens. By combining these two approaches, I will better understand P. aeruginosa localization mechanisms and will potentially elucidate aspects of the virulence of this important pathogen. In recent years, it has become accepted that bacteria, despite their small size, intentionally target proteins to specific locations within the cell. This organization is required for many basic cellular functions. Therefore, understanding the mechanisms by which pathogenic bacteria position proteins within the cell could provide insight into how these microorganisms cause disease and contribute towards the future development of vaccines or treatments. [unreadable] [unreadable] [unreadable]